The Effect Of Tangshenping On Renal Protection And RhoA/ROCK Signal Transduction Pathway In Diabetic Nephropathy KKAy Mice

With the rapid development of China’s social economy,the prevalence of Diabetes Mellitus(DM)has been rising in China,It can be seen clearly that the incidence of DM in our country has been rapidly expanded in the past 30 years,the prevalence rate is about 9.7%,that is,about 92 million adults are suffering from DM,and 20-30%of them will develop into Diabetic Kidney Disease(DKD)and eventually become the primary cause of end stage renal disease(ESRD).The development of Diabetic Kidney Disease is related to many factors.The main aspects can be summarized as the result of synergistic effects of congenitally genetic factors and living habits.The pathological mechanisms are oxidative stress,lack of insulin secretion,decrease of insulin sensitivity and so on,and the renal pathological changes are the destruction of glomerular filtration membrane,glomerular sclerosis and renal tubular epithelial cell vacuolar changes,interstitial fibrosis et cetera.One of the important mechanisms of renal interstitial fibrosis is the Epithelial-Mesenchymal Transition(EMT),and the study of EMT-related mechanisms such as signal transduction pathways has become a hotspot in recent years.RhoA/ROCK signaling pathway is involved in the EMT process of renal tubular epithelial cells,TGF-β molecule has the very strong fibrogenic effects on kidney,and can get involved in the proliferation of DKD mesangial cells and increase production of extracellular matrix,tubular epithelial mesenchymal transdifferentiation and apoptosis of podocytes and other pathological processes through a series of signal transduction pathways,aggravating the disease process of DKD.When TGF-β stimulates the RhoA molecular,RhoA can be activated to further act on its downstream moleculars,Rho-associatedcoiled-coilproteinkinase(ROCK).There are two subtypes of ROCK,ROCK1 and ROCK2.ROCK1 is widely expressed in human body.When RhoA stimulates ROCK1,its molecules initiates the 854 serine and 697 threonine phosphorylation and makes them activated,causing EMT,then the a-SMA and E-cadherin expression are increased.In the EMT process,Rho activation is a critical step,high glucose or TGF-β1 can activate the RhoA/ROCK signaling pathway,and in the TGF-β1-induced high expression of a-SMA during EMT process,Rho plays a central role.In diabetic model mice,studies have found that the activation of RhoA/ROCK signaling pathway can lead to a decrease in E-cadherin proteinexpression,leading to the decrease of cell-to-cell adhesion.The reduced intercellular adhesion makes epithelial cells more easily shift to the renal interstitial,changes the type and become something different,namely transdifferentiation,and it changes into myofibroblast.Y27632(ROCK specific inhibitor)can down regulate the activity of ROCK molecule,and promote the formation of cell adhesion and cell adhesion-related molecules between adjacent renal tubular epithelial cells.Through this mechanism to repair the cell adhesion between renal tubular epithelial cells and inhibit or even reverse the process of transdifferentiation.RhoA/ROCK pathway is involved in cell adhesion and migration,proliferation,apoptosis,transcription of target genes,regulation of cell proliferation cycle,epithelial mesenchymal transition and the rearrangement of actin cytoskeleton.Based on the understanding of the elderly on DKD proposed,my mentor came up with the"consumptive kidney disease" scientific hypothesis,As the development of DKD,the heat,qi stagnation,phlegm,blood stasis and toxin intercross in the kidney,blocking the circulation of qi and blood,causing the" Renal structure" damage,and the structural damage of the kidney will eventually lead to the function of the kidney damage or even failure that is,"Renal function" disorders,so " Renal structure "and" Renal function" are both damaged,in the long run,causes the "consumptive kidney disease"."consumptive kidney disease " not only summed up its pathogenesis,but also describes the final state of the disease.Under the instruction of "consumptive kidney disease" scientific hypothesis,our scientific research team use Tangshenping which is a prescription based on the " consumptive kidney disease"scientific hypothesis,to perform a large number of experiments to prove the hypothesis under the huge support by the National Natural Science Foundation of China.Immunohistochemistry,in situ hybridization histochemistry,and Western blotting were used to study the related proteins and genes of renal tubular epithelial cell transdifferentiation.The whole animal experiment was used to clarify the effect of Tangshenping on diabetic kidney disease.This study uses DKD KKAy mice and mouse renal tubular epithelial cells as the research object,by immunohistochemistry,in situ hybridization and Western blotting method,to further explore the influence of Tangshenping on renal protection during DKD and on EMT related the key molecules in RhoA/ROCK signaling pathway and provide more experimental evidence for the scientific clarification of " consumptive kidney disease " scientific hypothesis.Objective:The animal experiment with DKD KKAy mice,the pharmacodynamics effects and oxidative stress influence of Tangshenping on DKD KKAy mice.Detecting the protein and mRNA expression of TGF-beta 1,RhoA,ROCK1,alpha-SMA,E-Cadherin in renal tissues of mice by immunohistochemistry and in situ hybridization histochemistry;cell culture experiments using mouse renal tubular epithelial cells,the positive control drug is irbesartan and RhoA/ROCK specific inhibitor Y27632,through the MTT experiment to clarify the effects of Tangshenping on glucose stimulated renal tubular epithelial cell proliferation level,the application of Western blotting is to detect the expression of RhoA,ROCK1,alpha-SMA,E-Cadherin in glucose stimulated renal tubular epithelial cell under the treatment of Tangshenping drug-containing serum.Methods:1.Pharmacodynamics of Tangshenping on DKD KKAy Mice.The normal control group was 10 female C57BL/6J mice.Sixty female 10-week-old KKAy mice were induced with KK feed for 10 weeks.If the blood glucose ≥16.7mmol/L,24h urine protein>0.4mg,then DKD animal model succeed.KKAy mice were randomly divided into model group,irbesartan group and small,middle and high dose group,and treated with the corresponding medicine.Recording the general condition of mice and the body weight was used to calculate for 24h protein of mice in all experimental groups.All mice’s blood samples were taken at twenty-sixth weeks via taking out their eye balls,and biological data such as blood glucose,triglyceride(Triglyeride,TG),creatinine(Serum creatinine,Scr)and urea nitrogen(Blood urea,nitrogen,BUN);weight of mice’s kidney were harvested after that,then to calculate kidney weight/body weight ratio;mouse kidney were paraffin buried,After slicing,used HE,Mallory and PAS staining to analyse mice’s kidney pathological and morphological changes.2.Serum levels of malondialdehyde(MDA),nitric oxide(NO)and superoxide dismutase(SOD)were measured by automatic biochemical analyzer(colorimetric method).3.Immunohistochemistry:The expression levels of TGF-β1,RhoA,ROCK1,a-SMA and E-cadherin were detected in mouse renal tissue.4.In situ hybridization histochemistry:The expression of TGF-β1,RhoA,ROCK1,α-SMA and E-cadherin mRNA were detected in mouse renal tissue.5.Western blotting:The expression of RhoA,ROCK1,α-SMA and E-cadherin were detected in renal tubular epithelial cells.6.The experimental datas were analyzed by SPSS 22.0 software.Results:1.Effects of Tangshenping on Pharmacodynamics of DKD KKAy MiceCompared with the model group,the general condition of mice in the small,middle and high dose groups were improved,and the body weight was decreased in different degrees.After 16 weeks of treatment,the body weight of the mice in high dose group was significantly reduced(P<0.01),the kidney weight/body weight ratio was significantly reduced(P<0.01),4h protein of mice was significantly reduced(P<0.01),the content of serum BUN was significantly decreased(P<0.01),the content of TG and Scr decreased(P<0.05).The glomerular basement membrane and tubular basement membrane is slightly thickened in middle dose group and high dose group of Tangshenping.Renal pathological changes are definitely restrained.2.Effects of Tangshenping on Oxidative Stress in DKD KKAy MiceCompared with the model group,the levels of NO and SOD in the serum were significantly increased(P<0.01),while the content of MDA was significantly decreased(P<0.01)in all the Tangshenping groups.3.Effects of Tangshenping on expression of TGF-β1,RhoA,ROCK1,α-SMA and E-Cadherin in renal tissue of DKD KKAy MiceImmunohistochemistry showed that the expression of TGF-β1,RhoA,ROCK1 and α-SMA in renal tubular epithelial cells of normal control group was significantly lower than that of model group(P<0.01).Compared with model group,the expression of TGF-β1,RhoA,ROCKI and a-SMA protein was significantly decreased in the epithelial cells(P<0.01)except the low dose group.Compared with the model group,the expression of E-cadherin protein in the cytoplasm of renal tubular epithelial cells in the normal control group and other groups(except low dose group)was significantly higher than that in the model group(P<0.01).Cadherin protein expression were significantly increased(P<0.01),and the middle dose and high dose group was the most obvious among all Tangshenping groups.4.Effects of Tangshenping on the expression of TGF-β1,RhoA,ROCK1,α-SMA and E-Cadherin mRNA in renal tissue of KKAy mice with DKDIn situ hybridization showed that the expression of TGF-β1,RhoA,ROCK1 and a-SMA in renal tubular epithelial cells of normal control group was significantly lower than that of model group(P<0.01).Compared with model group,the expression of TGF-β1,RhoA,ROCK1 and a-SMA protein was significantly decreased in the epithelial cells(P<0.01)except the low dose group.Compared with the model group,the expression of E-cadherin protein in the cytoplasm of renal tubular epithelial cells in the normal control group and other groups(except low dose group)was significantly higher than that in the model group(P<0.01),Cadherin protein expression were significantly increased(P<0.01),and the middle dose and high dose group was the most obvious among all Tangshenping groups.5.Effects of Tangshenping on expression of RhoA,ROCK1,a-SMA and E-cadherin in renal tubular epithelial cells stimulated by high glucose in vitroWestern blotting showed that RhoA,ROCK1 and a-SMA were significantly lower in the normal control group than those in the high glucose group(P<0.01).Compared with the her group,the expression level of RhoA,ROCK1 and a-SMA protein in the middle dose group and high dose group of Tangshenping were significantly lower(P<0·01),the expression level of E-Cadherin in normal control group was significantly higher than the high glucose 1 group(P<0.01);compared with the high glucose group,the E-Cadherin protein expression in middle dose group and high dose group of Tangshenping increased significantly(P<0.01).Conclusion:1.Tangshenping can improve the general condition of DKD KKAy mice,reduce the body weight,kidney weight/body weight ratio,reduce the 24h urine protein quantification,and reduce the kidney pathological damage of KKAy mice.The effects above are dose related.2.The level of NO,SOD in DKD KKAy mice could decrease by Tangshenping to regulate the level of oxidative stress so as to protect the renal function.3.The expression of TGF-β1,RhoA,ROCK1,a-SMA protein and mRNA in renal tissue of DKD KKAy mice decreased while the expression of E-cadherin protein and mRNA increased by the use of Tangshenping,so as to restrain the EMT in DKD KKAy mice.4.The expression of RhoA,ROCK1 and a-SMA protein in renal tubular epithelial cells stimulated by hyperglycemia in vitro can be decreased by Tangshenpin medicine containing serum and increase the expression of E-cadherin protein to inhibit the EMT of renal tubular epithelial cells.TangshenPing can improve the general status of DKD KKAy mice,and its effect on the renal protection and reverse renal tubular epithelial cell transdifferentiation in DKD KKAy mice may be related to down-regulation of oxidative stress and regulation of RhoA/ROCK signaling pathway,In vitro cell experiments have also drawn the same conclusion.All above provide plenty new scientific material for the core theory of "consumptive kidney disease"hypothesis.